The present invention relates to high performance liquid chromatography.
In high performance liquid chromatography (HPLC), a liquid usually has to be provided at a very controlled flow rate (e.g. in the range of microliters to milliliters per minute) and at high pressure (typically 200-1000 bar, and beyond up to currently even 2000 bar, at which compressibility of the liquid becomes noticeable). Piston or plunger pumps usually comprise one or more pistons arranged to perform reciprocal movements in a corresponding pump working chamber, thereby compressing the liquid within the pump working chamber(s). In fluid dynamics and hydrometry, the volumetric flow rate (referred to herein as flow rate) is the volume of fluid which passes through a given surface per unit time, usually measured at the point of detection.
A liquid chromatography pumping system is described in EP 0309596 B1 by the same applicant, Agilent Technologies, depicting a pumping apparatus comprising a dual piston pump system for delivering liquid at high pressure for solvent delivery in liquid chromatography.
Modern LC-systems see changing requirements. In the interest to increase peak capacity (i.e. the total number of peaks per time interval) several parameters are optimized, such as smaller size of packing material, smaller inner diameter columns, faster linear speed of solutes during separation, faster compositional gradients, longer separation beds, etc. Most of these developments have in common that they increase the pressure drop needed to drive the liquid through the system.
HPLC systems often are operated in so-called gradient mode, wherein e.g. for reversed phase chromatography the organic content is ramped over time, or for ion exchange chromatography the salt content is ramped over time. Especially in proteomics most applications are based on water/acetonitrile gradients.
An analytical protocol for running a defined analytical process is called the “method”. In the analytical protocol—or method—for a gradient separation, the gradient is specifically defined as a composition change (e.g. % B over time), while the flow rate is kept constant over the major part of such a method.
Increasing the pressure drop across a column may bring the method execution close to limits of the technical hardware. Modern LC-Systems already are designed to leverage all pressure capabilities of the given setup. To prevent overstress to certain components the system is often equipped with pressure sensing having shut-off features, which are designed to terminate operation before the hardware is damaged.
Column temperature might be increased in a quest to gain more headspace in pressure by reducing viscosity, thus reducing nominal pressure drop of a given column.
Chromatographic columns filled with smaller particles are susceptible to clogging, which goes back to either solid material from dirty samples injected or particles and abbreviates that might stem from seals or valves.
For chromatographers it is good laboratory praxis not to use all pressure reserve available right from start. Minor clogging of the column would then raise pressure above the shut-off limit. Often about 10-20% of the available maximum pressure range remains unused when optimizing the method, in particular to avoid measurement failures caused from temporarily exceeding shut-off limits.